EISEVIER European Journal of Pharmacology

Molecular Pharmacology Section 291 (1995) 281-289

molecular pharmacology

Characterisation of a recombinant P,, purinoceptor

Joseph Simon a*‘, Tania E. Webb a, Brian F. King b, Geoffrey Burnstock b, Eric A. Barnard a,* a Molecular Neurobiology Unit, Royal Free Hospital School of Medicine, Rowland Hill Streer. London NW3 2PF, UK

b Department of Anatomy and Developmental Biology and the Centre for Neuroscience, University College London, Gower Streer, London WClE 6BT, UK

Received 12 July 1995; accepted 21 July 1995

Abstract

We have previously cloned a cDNA encoding a G-protein-coupled Pr purinoceptor from chick brain and designated this as a P2v, putinoceptor (Webb, T.E., J. Simon, B.J. Krishek, A.N. Bateson, T.G. Smart, B.J. King, G. Bumstock and E.A. Barnard, 1993, FXBS Lett. 324, 219). Here, we describe the further characterisation of this recombinant receptor expressed in both simian kidney endothelial (COS-7) cells and Xenopus oocytes. In transfected COS-7 cell membranes, the recombinant receptor showed a high level of expression

@“ax = 7.9 f 2.2 pmol [35SklATPcuS bound/mg protein) and affinity (Kd = 6.6 f 0.3 nM). In these COS-7 cells, the activation of the implanted purinoceptor induced a suramin-sensitive formation of inositol 1,4,5trisphosphate (1,4,5InsP,). Upon expression in Xenopus

oocytes, ATP was the only natural nucleoside triphosphate to elicit a Ca2+ -activated chloride current. The P2 purinoceptor antagonists suramin and Reactive Blue-2 were both able to inhibit this evoked current. Utilizing both expression systems, the binding affinity profile and the functional pharmacological profile of the agonists, the common series found was: 2-methylthioATP (2-MeSATP) 2 ATP > ADPp S > ADP. These two agonist series and the lack of activity of adenosine, cr. /3-methyleneATP ( a,P-meATPI, 3’-O-(4benzoyl)ben- zoyl-ATP (Bz-ATP) and UT’P, together confirmed that this receptor is a specific subtype of the P2v purinoceptors.

Keywords: Brain, chicken; G-protein-coupled receptor; Recombinant P rv, purinoceptor; Xenopus oocyte expression; COS-7 cell, transient expression;

Inositol 1,4,5-t&phosphate, formation

1. Introduction

Receptors for extracellular ATP and other nucleotides

are designated as Pz purinoceptors, distinct from the P, purinoceptors specific for adenosine (Bumstock, 1978). P, purinoceptors have been further divided into subtypes on the basis of the selectivities for various nucleotide agonists

and their congeners. The two major and best characterised subtypes of ATP receptors, suggested by Bumstock and Kennedy (1985), are the Pzx and P,, purinoceptors. The Pzx purinoceptors are members of the transmitter-gated ion channel superfamily, containing a non-selective cation channel, mediating fast excitatory neurotransmission and usually responding both to ATP and the P,,-selective

l Corresponding author. Tel.: 44 (OJ171 795 0500, ext. 5445; fax: 44 CO)171 431 1973.

’ Present address: Glaxo Institute of Applied Pharmacology, Depart-

ment of Pharmacology, University of Cambridge, Tennis Court Road,

Cambridge CB2 lQJ, UK.

0922-4106/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved

SSDI 0922-4106(95)00115-8

agonist (Y, P-methyleneATP ( (Y, P-meATP1 (as reviewed by Bean, 1992 and Edwards and Gibb, 1993). Recently two cDNAs encoding subtypes of the Pzx purinoceptors have been cloned: the receptor structures show major differences from the other ligand-gated cation channels

(Valera et al., 1994; Brake et al., 1994). The application of ATP and ADP at many sites pro-

duces far slower responses via PzY purinoceptors (at which (Y , P-meATP is relatively inactive), suggesting a metabotropic signalling pathway. The involvement of G-

proteins in this cascade has been established (Dubyak, 1991), thus identifying the PzY purinoceptor as a member of the superfamilies (Barnard, 1992) of G-protein-coupled

receptors. PzY purinoceptors have been found in many locations (reviewed by Bumstock, 1993), including vascu- lar and visceral smooth muscles, epithelial and endothelial cells, astrocytes and turkey erythrocytes. The binding to the Pzu purinoceptor of ATP or its analogue 2-methyl- thioATP (2-MeSATP) leads, in the cases studied so far, to the generation of inositol 1,4,.Ctrisphosphate (1,4,5InsP,) (Dubyak, 1986) which, in turn, is linked to the mobilisa-

282 J. Simon et al . / European Journal of Pharmacology Molecular Pharmacology Section 291 (1995) 281 289

tion of intracellular calcium (Forsberg et al., 1987; Boyer et al., 1989). Exceptionally, in astrocytes stimulation of a P2v purinoceptor leads to an increase in the levels of eicosanoids (Bruner and Murphy, 1990).

Further subtypes within the G-protein-coupled class of P2 purinoceptors have been proposed, based on the relative rank orders of potency at them of ATP analogues. These include the P2T receptor on the platelets and megakary- ocytes, at which ADP is the preferred agonist but ATP is an antagonist (Gordon, 1986; Murgo et al., 1994). The action of ADP and its derivatives at these sites has been reported to involve an inhibition of adenylate cyclase (Cristalli and Mills, 1993). Another receptor subtype, the P2u or nucleotide receptor, has been defined to accommo- date the observation that, in certain tissues or cells, UTP is as active as ATP and the P2y-selective agonist 2-MeSATP has much less affinity for these sites (Dubyak, 1991; O'Connor et al., 1991). The P2u purinoceptor, like the P2Y, is thought usually to generate 1,4,5InsP 3, but a P2u subtype in hepatocytes has also been reported to be negatively coupled to adenylate cyclase by a pertussis toxin-insensi- tive Gi-protein (Keppens, 1993).

We have recently cloned a cDNA encoding a novel P2 purinoceptor (clone ChR803), abundant in the chick brain (Webb et al., 1993). Concurrently, a recombinant P2u p u r i n o c e p t o r has been desc r ibed from the neuroblastoma/glioma cell line NG108-15 (Lustig et al., 1993). Both of these have the seven-hydrophobic-domain structure of the G-protein-coupled receptors. While there have been indirect indications for the presence of P2 purinoceptors of the G-protein-coupled receptor type in the central nervous system (Fournier et al., 1990; Bruner and Murphy, 1990), very little is known of the function and pharmacology of brain P2 purinoceptors. We report here a characterisation of a recombinant P2 purinoceptor derived from brain mRNA (Webb et al., 1993), expressed in simian kidney endothelial (COS-7) cells and in Xenopus oocytes. These studies designate it as a specific PzY sub- type, termed P2Y1-

2. Materials and methods

2.1. Materials

[3SS]3'-Deoxyadenosine 5'-O-(l-thio) triphosphate ([35S]dATPoeS; 1400 Ci/mmol), dATPoeS, ATPolS and [3H]UTP (14 C i /mmol) were from New England Nuclear. Adenosine, ATP, c~,/3-methyleneATP (~,/3-meATP), 3'- O-(4-benzoyl)benzoyl-ATP (Bz-ATP), ADP, ADP/~S, UTP, Reactive Blue-2 (RB-2), chloroquine, theophylline and trypsin were all from Sigma. 2-MethylthioATP (2- MeSATP) and 2-methylthioADP (2-MeSADP) were from Research Biochemicals International and suramin from Bayer Pharmaceuticals (UK). The t.-ribose enantiomer of /3,y-methyleneATP (L-/3,y-meATP) was kindly donated by

Fisons Pharmaceuticals (UK). All tissue culture media and reagents were from Gibco. Other chemicals were from BDH (UK). Buffer A has the composition: 50 mM Tris/1 mM EDTA/1 mM EGTA, adjusted to pH 7.4 with HC1, and also contains (as protease inhibitors) 1 mM benzami- dine, 0.1 mM phenylmethylsulphonyl fluoride, 0.01% baci- tracin, 0.001% soybean trypsin inhibitor and 40 kallikrein inhibition units of aprotinin (Bo et al., 1992).

2.2. Cell culture and transfection

COS-7 cells were grown in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum, 2 mM glutamine and 100 units/ml penicillin and 0.1 mg/ml streptomycin, at 37°C under a C O J a i r atmosphere (8%/92% v / v ) for 18-24 h prior to transfection. The expression constructs (pSG5/803 and pcDNAIAmp/803) were generated by subcloning the entire EcoRI insert of the ChR803 cDNA clone (Webb et al., 1993) into the reciprocal site of the eukaryotic expression vector pSG5 (Stratagene) and of pcDNA I Amp (Invitrogen). The resul- tant plasmid was transfected into COS-7 cells by the DEAE-dextran method (McCutchan and Pagano, 1968). Control COS-7 cells were transfected with the vector alone, but were otherwise treated as the construct-trans- fected cells. The transfected cells were cultured for 72 h and harvested in buffer A.

2.3. 1,4,51nsPs measurement

At 72 h post-transfection, COS-7 cells were washed 3 times with Dulbecco's modified Eagle medium and care- fully scraped from the dishes. The intact cells (5 × l0 s per 400 p.1 medium) were incubated with 1 or 10 / ,M agonist in Dulbecco's modified Eagle medium for 5 min at room temperature. Some aliquots of the cells were pre-incubated with 100 /,M suramin for 30 rain at room temperature prior to the addition of the agonist. The reactions were terminated by addition of 0.2 volume of ice-cold 20% perchloric acid and kept on ice for 20 min. The disrupted cells were then centrifuged ( 1 2 0 0 0 × g , 15 min) in a microcentrifuge at 4°C. The supernatants were transferred into siliconised polypropylene tubes and neutralised with KOH. The 1,4,5InsP 3 levels in the supernatants were measured using the [3H]D_myo_inositol 1,4,5-trisphosphate assay system (Amersham), according to the manufacturer's protocol.

2.4. Preparation of the crude membrane fraction

The harvested cells in buffer A were freeze-thawed and further disrupted by homogenisation with a Ultra-Turrax 3-25 homogeniser (2 × 15 s, setting 5, cooling the suspen- sion for 1 rain between pulses). The membranes were collected by centrifugation at 12000 × g, 30 min in a microcentrifuge at 4°C. The supernatant was discarded, the

J. Simon et al. / European Journal of Pharmacology - Molecular Pharmacology Section 291 (1995) 281-289 283

membranes were resuspended in buffer A (1 ml) by pass- ing through a 21-gauge sterile needle and incubated on ice (30 min) to chelate endogeneous divalent cations, destroy labile endogeneous ligands and inactivate traces of pro- teases. The membranes were then centrifuged and washed with buffer A twice. The pellet was resuspended in buffer A to give a protein concentration (Bradford, 1976) of 0.1-0.2 m g / m l and frozen in liquid N 2 before storage at - 70°C.

2.5. Radioligand binding assays

Aliquots (0.5 ml final volume) of the freeze-thawed membrane fraction containing 5-10 p.g protein in buffer A were incubated with the radioligand for 1 h at room temperature. [35S]dATPa S was used at a final concentra- tion of 7 nM ( ~ Kd), or 0.5-40 nM in the saturation experiments in the absence or presence of 100 /xM unla- belled 2-MeSATP (to define the non-specific binding).

[3H]UTP was at a saturating concentration of 40 nM, in the absence or presence of 100 ~ M unlabelled UTP. All assays were terminated by rapid filtration through G F / C glass fibre filters (pre-soaked in 20 mM sodium pyrophos- phate) and the filters were immediately washed with 3 × 5 ml of iced 50 mM Tris /HCl (pH 7.4) on a Millipore vacuum manifold. Filters were dried under an infra-red lamp and their radioactivity was determined using Op- tiphase 'HiSafe' II (LKB) scintillant, at a counting effi- ciency routinely of 95% for 35S and 60% for 3H.

2.6. Electrophysiology

Xenopus oocytes were cytosol-injected with cRNA (transcribed in vitro from pSG5/803 capped and polyadenylated (Webb et al., 1993); ~ 80 ng/oocyte) or nuclear-injected with cDNA (pCDNA-IAmp/803; ~ 10 ng/oocyte), using either folliculated or defolliculated oocytes. The defolliculation procedure used is described

400] " A

300] 135S IdATPctS [3HIUTP .'~', 6

,-~ 4' 200 ~o

1 1 ) 0 ~ m ~ 2

pSG5/803 Control pSG5/803 Control

B

0 lb 30 F (nM)

1.0"

0.8"

0.6

c

0.4-

0.2 • , • , • , • , . , . 0 1 2 3 4 5 6

B (pmol/mg protein)

1 0 0 ~ D

o I i

-1 l 9 7 5 -3

loglDisplacerl , (M)

[] d A T P ~ S

• 2 -MeSATP

o ATP

zx ADP

• Suramin

v RB-2

• UTP

• L-13,y-meATP

Fig. 1. Specific binding of potential agonist radioligands to COS-7 cell membranes transiently expressing the recombinant purinoceptor. A: Membranes of pSG5/803-transfected or control-transfected COS-7 cells (10 p.g protein per tube) were assayed with 40 nM [35S]dATPotS (as marked) in the absence of (filled bars) or presence of 100/zM unlabelled 2-MeSATP (open bars) or of 100/xM unlabelled RB-2 (lightly shaded bar). Alternatively, where marked, 40 nM [3H]UTP was used in the absence of (filled bar) or presence of 100/xM unlabelled UTP (hatched bar). The S.E.M. range is shown for each mean value. B: Saturation isotherm for [3~S]dATPaS-specific binding. Non-specific binding was in the range of 10-30% of the total binding, up to saturation (not shown). C: Scatchard plot. The points represent the values obtained in one of the six independent transfection experiments with [35S]dATPctS, which all gave extremely similar results. Analysis (see Materials and methods) yielded (mean + S.E.M.) K d = 6.6 5:0.3 nM, Bm~ x = 7.9 + 2.2 pmol /mg protein. The theoretical curve (B) and line (C) are generated from these parameters. Hill plots (not shown) gave a mean n H of 1.01. D: Competition of purinoceptor-active ligands for specific [35S]dATPaS sites. Each ligand was added with the [35S]dATPaS (6.5 nM) and.incubated with the membrane fraction of the transfected cells for 1 h at room temperature. Points represent the values obtained in one of the four independent transfections, all of which gave extremely similar results. The curves are theoretical (n H = l) for the one-site binding model (which always gave the best fit) and the parameters are given in Table 1 for the recombinant receptor in COS-7 cells.

284 J. Simon et al./ European Journal of Pharmacology - Molecular Pharmacology Section 291 (1995) 28l 2~9

elsewhere (Ziganshin et al., 1995). No significant differ-

ences were detected in the potency of ATP for P2YI purinoceptors expressed by either route and for folliculated vs. defolliculated oocytes. Two-electrode voltage clamp

recording was performed using an Axoclamp 2A (Axon

Instruments) amplifier. Dual-impaled oocytes were used if

their resting potential was more negative than - 4 0 mV

and input resistance greater than 0.5 M.Q. All drugs tested

were dissolved in amphibian Ringer solution (without Mg 2÷, as in Webb et al., 1993) and the recording chamber

(bath volume 0.5 ml) was superfused at a rate of 5 m l / m i n . A minimum period of 20 min wash with Ringer

solution followed each application of drug to overcome any desensitisation. The endogeneous adenosine receptor

of Xenopus oocytes (Lotan et al., 1986) was not detectable

in most batches of oocytes, so an inhibitor thereof was not

then used. It was found to be present in some batches, and then 100 /xM theophylline was added to inhibit it com-

pletely. It was verified that the response of the expressed P2Y receptor to 2-MeSATP was quantitatively identical with and without the addition of theophylline. The potency

of ATP (and 2-MeSATP) in the present study was consid- erably higher than found in an earlier pilot expression

study (Webb et al., 1993) of the recombinant P2Y~ purinoceptor. We attributed this change, in part, to a difference in the earliest batches of oocytes used and a variability in the activity of the Ca2+-activated chloride

channel as described by Boton et al. (1990). The ECs0 values for ATP and its analogues described here accord

well with their ECs0 values at the recombinant P2Y purinoceptor from turkey brain in stimulating 1,4,5InsP 3 formation (Filtz et al., 1994).

2.7. Data analysis

All experiments were carried out in triplicate and were

repeated at least 3 times. Data are expressed as overall

mean + S.E.M. All the binding data were analysed using the EBDA-LIGAND computer program (Biosoft, Cam-

bridge, UK). In each case alternative one-site or two-site binding models were used; the appropriate model was

selected on the basis of an F-test on the weighted residual

sums of squares and standard errors (Munson and Rod-

bard, 1980). Data from dose-response curves of electro- physiological measurements were normalised to the maxi-

mum response obtained to each drug for each oocyte, to

overcome differences in oocyte size and chloride channel

density. ECs0 values were estimated from Hill plots and expressed as mean _+ S.E.M. for 3 or more determinations.

The activity of ATP analogues was compared against the

response evoked by 1 tzM ATP ( = EC~00) which was taken as 1.

3. Results

3.1. Transient expression o f the recombinant Per purinoceptor in COS-7 cells

[35S]dATPaS, an ATP-derived radioligand which has

advantages for labelling PzY purinoceptors (Simon et al., 1995), bound to COS-7 cells expressing the receptor; this binding was completely abolished by the addition of a large excess (100 /~M concentration) of the P2v-selective agonist 2-MeSATP (unlabelled), or of the antagonist RB-2

Table 1 Potencies of purinoceptor-active ligands at the chick brain native and the recombinant purinoceptors

Ligand Recombinant COS-7 expressed in Xenopus Native brain Pzv cells g i ( n M ) oocytes ECso (nM) purinoceptors a Ki (nM)

2-MeSATP 69 4- 23 10 + 1 34 + 8 dATPo~S 23 + 7 nd 17 + 2 ATPo~S 63 + 22 nd 32.0 ___ 9 ATP 48 + 13 155 + 50 47.8 4- 4 ADP 171 __+ 19 258 + 40 b 530 + 183 2-MeSADP 326 + 176 nd 170 + 38 ATPyS 52 + 17 nd nd ADPb 91 + 18 ~ 150 c nd Suramin 1592 + 206 230 + 80 d 1052 4- 244 RB-2 944 + 201 580 4- 130 d 1472 4- 278 UTP 6077 + 327 > 10000 > 10000 Adenosine > 10000 > 10000 > 10000 L-/3,y-meATP > 10000 > 10000 > 10000

The inhibition constant (K,) for each ligand was computed from the competition data (see examples in Fig. 1D) using the Kj values observed of 13.3 nM (native) or 6.6 nM (recombinant receptor) for [35S]dATPaS binding. The ECsl ) or IC5~ ~ values were derived from dose-response curves (examples in Fig. 3) measured on the injected Xenopus oocytes. All values are expressed as the mean + S.E.M. of at least three independent determinations. ~ For comparison, values obtained on brain membranes from the newly-hatched chick (Simon et al., 1995) are also shown, b Measured for the first phase (see Fig. 3) only; nd, not determined. ~ Dose-response curves not determined but based on equipotency to ATP (see Table 2). a Represents an 1C5o value for antagonists, suramin and RB-2.

J. Simon et al. / European Journal of Pharmacology - Molecular Pharmacology Section 291 (1995) 281-289 285

(Fig. 1A). Neither [35S]dATPaS (Fig. 1A) nor [3H]UTP (which recognises the P2u subtype) gave any specific binding to the pseudo-transfected (pSG5 vector alone) COS-7 cell membranes, showing the absence of any inter- feting level of native P2 purinoceptors in these cells. In addition, no specific binding of [3H]UTP (Fig. 1A) or of [3H]a,/3-meATP (data not shown) to the pSG5/803-trans- fected cell membranes was found. Furthermore, a Northern blot of total RNAs extracted from COS-7 cells transfected with the receptor cDNA/vector construct pSG5/803 (Webb et al., 1993) showed a hybridising species of 1.7 kb when it was hybridised under conditions of low stringency with a labelled 1.1 kbp EcoRI fragment, containing the coding region of that cDNA (data not shown). No signal was seen in the case of the control, pseudo-transfected cells (data not shown). These results indicated that COS-7 cells do not express an endogenous receptor of this type and that, post-transfection, they abundantly express the receptor.

The binding of [35S]dATPaS to the membrane prepara- tion containing the recombinant receptor was saturable (Fig. 1B and C). The data were best fitted by a single binding site model (see Materials and methods). The anal- ysis gave a dissociation constant (K d) of 6.6 __+ 0.3 nM for this high affinity binding site (maximal number of binding s i tes , Bma x: 7.9 + 2.2 pmol/mg protein). The pseudo-Hill coefficient (n H) for these sites was 1.01 + 0.05, indicating a single set of high affinity sites (Hill plot not shown).

Competition with the [35S]dATPaS binding to the re- combinant receptor was investigated for a range of com- pounds active on purinoceptors (Fig. 1D). dATPctS, ATPaS, ATP and 2-MeSATP all competed with high affinity at these binding sites, while ADP, 2-MeSADP and ADP/3 S were rather less active. However, other purinocep- tor agonists such as L-/3,~/-meATP (strongly P2x-selective: Cusack et al., 1987), UTP or adenosine did not signifi- cantly displace [35S]dATPt~S binding at 10 -6 M concen- trations (Fig. 1D). Suramin (Dunn and Blakeley, 1988) an antagonist for P2 purinoceptors in general, and RB-2, an antagonist for P2v purinoceptors over a narrow concentra- tion range (Burnstock and Warland, 1987), each competed with the binding of [35S]dATPctS to these sites, with IC5o values around 2 /xM (Fig. 1D). The inhibition constants ( g i) determined from such data (Table 1, column 2) show that the rank order of affinity of the purinergic ligands tested here, in displacing [35S]dATPctS binding from the recombinant receptor is: dATPaS > (ATP, ATPaS, ATPTS, 2-MeSATP) > ADPflS > ADP > 2-MeSADP > (RB-2, suramin)>> UTP, L-fl,~/-meATP, adenosine. (The ligands within parentheses do not show statistically signifi- cant difference in their affinities.) This potency order for binding to the recombinant P2 receptor from late-em- bryonic chick brain agreed with that established on brain membranes from the newly hatched chick for the native P2 purinoceptors, in which P2Y types greatly predominate (Table 1).

A

B

C

ATP, 10~M ATP ATP

uninJected sterile water P~; purlnoceptor

2-MeSATP ATP ADP AMP Ad ct,13- I~,y~meATP

3 mln

2-MeSADP ATP ADP~ ATPaS Bz-ATP UTP

3 rain

D RB2 10 ~ 3x10 "6 l0 "6 3xi0 -'s I0"4M A ~ 3x io ~

3 rain

Fig. 2. Responses by Xenopus oocytes to the supeffusion of ATP and other nucleotides. Each agent shown was applied at a concentration of 10 .6 M, for 1 min. A: Responses to ATP from an uninjected oocyte or a

water-injected oocyte or an oocyte injected with cRNA transcribed from pSG5/803. B and C: Relative responses to purine derivatives by another two oocytes expressing the purinoceptor (Ad, adenosine; ct,/3-, ot,/3- meATP; BzATP, 3'-O-(4-benzoyl)benzoyl-ATP). D: RB-2 (at the molar concentrations marked) antagonised the responses to ATP (3 × 10 -7 M) on an oocyte expressing the recombinant purinoceptor. The response to ATP after RB-2 block is partly recovered after 1 h wash, although the onset and offset rates of the oscillatory currents were significantly slower. Note: The response to ATP was close to its maximum in all oocytes tested (cf. Fig. 3). In panel A, the small downward deflections monitor the input conductance by periodically applied voltage steps ( - 10 mV, 1 s).

3.2. Expression of the receptor in Xenopus oocytes

ATP evoked large, slowly developing, chloride currents (Ia.ca) with a secondary oscillatory phase, characteristic of G-protein-coupled receptor responses in cRNA or expres- sion construct-injected oocytes (Landau and Blitzer, 1994). It failed to elicit any inward current in either uninjected oocytes or oocytes injected with sterile water (Fig. 2A). Of the naturally occurring purine and pyrimidine nucleotides tested, ATP was the only active nucleoside triphosphate at this P2 purinoceptor (Table 2). Inward currents evoked by a series of purinergic ligands at 10 -6 M concentration consistently yielded the order of activity; 2-MeSATP > 2- MeSADP > ATP = ADPflS > ADP > ATPaS >> AMP, with a,/3-meATP, /3,~/-meATP, Bz-ATP, UTP and adeno- sine inactive (Fig. 2B and C, and Table 2).

286 J. Simon et al./ European Journal of Pharmacology Molecular Pharmacology Section 291 (1995) 281-289

Table 2 50 Relative activities of nucleotides and adenosine at the recombinant purinoceptor expressed in Xenopus oocytes ~ "" 40

Agonist Activity n (at 1 /xM) ~ 30

ATP 1 3 ADP 0.65+0.08 6 ~ 20 AMP 0.09 + 0.09 3 "~" Adenosine 0 3 ~ 1 0 2-MeSATP 1.60 5:0.25 3 2-MeSADP 1.28 _+ 0.11 3 (2')dATP 0.23 1 ATPa S 0.32 + 0.09 4 ADP/3 S 1.04+0.13 3 a,/3-meATP 0 3 80 /3,y-meATP 0 3 .~ Bz-ATP 0 3 ~ ( UTP 0.03 + 0.03 3 ¢a4

CTP 0.01 _+ 0.01 3 GTP 0.07 + 0.07 3 ~

t t~ ITP 0.03 5:0.03 3

The activity of each compound was determined by comparing the ampli- "~ tude of evoked currents to the reponse to ATP (taken to be 1). Each compound were used at 1 /a.M which represented ECru0 for ATP. Experiments were carried out on three or more oocytes and the results expressed as a mean + S.E.M.

2 - M e S A T P e v o k e d an i n w a r d cu r ren t at a l ower th resh -

o ld c o n c e n t r a t i o n and p r o d u c e d m a x i m u m cur ren t s at l o w e r

c o n c e n t r a t i o n s t han d id A T P (Fig. 3). T he ECso for 2-

M e S A T P was low, 10.3 + 0 .14 nM, some 15-fold l ower

than tha t for A T P (Tab le 1). 2 - M e S A T P was a ful l agon i s t

at the P2Y] pu r inocep to r , bu t no t A T P (Tab le 2) no r A D P

pSG5

T

P2Y I/P SG5

B

r-----i Basal

1 ~tM 2-MeSATP

I ~tM ATP

1 ~tM 2-MeSATP + 100 ~M Suramin

[---] Basal

101xM 2-MeSATP

10~tM dATPctS

101xM ATPetS

10~tM ATPTS

P2Y1/pSG5

Fig. 4. A P2¥ agonist induces 1,4,5InsP3 accumulation in intact COS-7 cells transiently expressing the recombinant purinoceptor. The amount of 1,4,51nsP 3 synthesised is expressed as attomoles per cells+S.E.M. (3 independent determinations, each in triplicate). A: The cells were incu- bated with or without (basal) 1 /zM 2-MeSATP or 1 /a.M ATP for 5 min, or incubated with 100 /zM suramin prior to and during the 2-MeSATP treatment. B: The cells were incubated with one of the following ligands: 10 /xM 2-MeSATP, 10 /xM dATPaS, 10 /xM ATPo~S, 10 /xM ATPyS for 5 min.

> ' 0

~ E

%

100-

50-

/

I - - T 9 8

)

O - I I I I I

7 6 5 4 3

-log [agonistl

Fig. 3. Relationship between the agonist dose (logl0 of the molar concentration) and the cell response (inward current, as in Fig. 2) for oocytes expressing the P2 purinoceptor. Agonists: 2-MeSATP (0 ) , ATP (11), ADP (©). Each point shows the mean+S.E.M, for recordings on 3 oocytes (6 for ADP) The data are normalised with respect to the absolute maximum response obtainable in each oocyte (set at 100%) to remove the effect of variations in response between oocytes. Note that the absolute maximum responses differed between the 3 agonists as can be seen for the values at 1 /.tM of each agonist (see Table 2). The dose-response relationship for ADP was complex and gave the best fit to a biphasic curve as shown.

(Fig. 3). The m a x i m u m r e s p o n s e s to A T P and A D P were

a lways s ign i f i can t ly less than tha t to 2 - M e S A T P . T h u s in

t e rms of po tenc ies the o rde r is 2 - M e S A T P > A T P > ADP.

W i t h ADP, there were appa ren t ly two af f in i t ies (Fig. 3), to

be d i scussed be low. For the p r e d o m i n a n t set of A D P

b ind ing sites the ECso was a b o u t 1 .6-fold w e a k e r than that

o f A T P (Fig. 3; T a b l e 1). S u r a m i n (Tab le 1) and R B - 2

bo th a n t a g o n i s e d A T P r e s p o n s e s in a d o s e - d e p e n d e n t man-

ner ; su ramin s h o w e d a r eve r s ib l e a n t a g o n i s m whi le the

b l o c k i n g e f fec ts o f R B - 2 were no t ful ly r eve r s ib le (Fig.

2D).

3.3. Signalling properties o f the recombinant P2 Y purinoceptor

C O S - 7 cel ls e x p r e s s i n g the r e c o m b i n a n t pu r inocep to r

were also used to iden t i fy a s igna l l ing p a t h w a y for it. The

P2v-se lec t ive agon i s t 2 - M e S A T P e l ic i ted an app rox ima te ly

4-fold inc rease in the f o r m a t i o n o f 1 ,4 ,5InsP 3 in in tac t

C O S - 7 cel ls e x p r e s s i n g the r e c o m b i n a n t p u r i n o c e p t o r (Fig.

4A) . H o w e v e r , A T P was s o m e w h a t less ac t ive than 2-

M e S A T P . W h e n the cel ls were p r e - i n c u b a t e d wi th the P2

pu r inocep to r a n t a g o n i s t su ramin , the e f fec t o f 2 - M e S A T P

J. Simon et al. / European Journal o f Pharmacology - Molecular Pharmacology Section 291 (1995) 281-289 287

on the formation of 1,4,5InsP 3 was completely blocked. Control cells, transfected with the vector (pSG5) alone, gave no increase at all upon application of any of ligands tested (Fig. 4A and data not shown). ATPo~S or its deoxy derivative, dATPa S (used in the binding study), as well as ATPTS also stimulated in the formation of 1,4,5InsP 3, confirming their agonist properties at this recombinant receptor (Fig. 4B). These results show that this P2 purinoceptor, when transiently expressed in COS-7 cells, is coupled to accumulation of the second messenger 1,4,5InsP 3 via an appropriate G-protein.

4. Discussion

4.1. Identity of the recombinant receptor

We have previously cloned a cDNA encoding a protein of 362 amino acids, identified by its sequence as a novel member of the G-protein-coupled receptor main superfam- ily (Webb et al., 1993), being in a new family in the latter (Barnard et al., 1994). The receptor mRNA was found to be abundantly expressed in one-day-old chick and adult chicken whole brain (Webb et al., 1994). Initial studies on the functional expression of this protein in oocytes sug- gested that it is in the P2v family of the purinoceptors and it was termed P2Yl (Webb et al., 1993; Abbracchio and Burnstock, 1994). This brain P2Y receptor can be related to P2 purinoceptors recently characterized in chick and rat brain membranes by their characteristic ligand binding properties (Simon et al., 1995), confirmed also by puriner- gic ligand binding in autoradiography on chick brain sec- tions (J. Simon et al., unpublished results). These results showed that there is a very abundant population of sites with a P2Y pharmacology (Table 1, column 3); for exam- ple, the Bma x for dATPc~S bound with high affinity (Kd: 13.3 nM) is 37 pmol /mg brain membrane protein. The P2x receptor sites of the brain are not included in this total, since L-/3,3,-meATP (highly P2x-selective, as noted above) did not displace the radioligand binding (Table 1). Hence, it is P2v types which produce a very high abundance of high-affinity purinoceptors in the brain. Although several P2v subtypes in the brain could contribute to this total (but not P2u, since UTP was inactive at these sites: Table 1, column 4), the non-significant differences in the ligand affinities between brain and expressed receptor (Table 1) show that P2YI can account for the great majority of these binding sites. These binding sites are prominent in several brain regions, including piriform cortex, ectostriatum, cere- bellum, and some nuclei in the telencephalon and mesen- cephalon (J. Simon et al., unpublished). This correlates with the high abundance of the mRNA of the P2Y t receptor seen in the chick brain brain (Webb et al., 1993) and with its regional distribution there (Webb et al., 1994).

In the present study, we have found a specific phar- macology of this brain P2Y purinoceptor in recombinant

form, in terms both of the electrophysiological responses induced by ATP-related ligands and of the binding of such ligands to it when expressed in cultured non-neuronal cells. In the oocyte expression experiments, the ineffec- tiveness of Bz-ATP, UTP and ITP and the lesser potency of ADP, were also important in the assignment of this as a P2Y receptor subtype, distinct functionally from the P2u, P2z or P2T types as defined in the earlier literature (Gordon, 1986; O'Connor et al., 1991). For comparison, the recom- binant P2U receptor from the mouse by Lustig et al. (1993), when expressed by cell transfection, responds equally well to ATP and UTP and its mRNA was found prominently in several peripheral tissues but only weakly in the brain. The opposite is true (Webb et al., 1993) for the recombinant PRY receptor described here. In conclu- sion, then, this appears to be a major P2 receptor type present in the brain.

4.2. Relative potencies and affinities of the agonists

The rank order of functional potency, 2-MeSATP > ATP > ADP >> adenosine, L-fl,3,-meATP, for our oocyte expressed receptor (Table 1) is in accordance with the broad definition of a P2Y purinoceptor subtype (Burnstock and Kennedy, 1985). The same is true for the fuller series which was defined by the experiments on the affinity order of ligand binding, i.e. dATPaS > (ATP, ATPaS, ATPTS, 2-MeSATP) > ADP/3S > ADP > 2-MeSADP > (RB-2, suramin)>>UTP, L-/3,3,-meATP, adenosine (Fig. 1D, Table 1) and this was generally the same for the native chick brain and the recombinant P2Y purinoceptors ex- pressed in COS-7 cells. ATP and 2-MeSATP showed equal affinity for the recombinant receptor and also for the brain membrane receptors, but 2-MeSATP is 15 times more potent than ATP in evoking the current response in oocytes. A possible artefact causing this difference, due to a hypothetical differential ecto-nucleotidase destruction of these nucleotides, was considered. In the binding assays, nucleotide hydrolysis was discounted because (a) mono- and divalent cations were omitted and chelating agents were present, conditions used because they had been shown to inhibit completely the ATPases of such membranes (Bo et al., 1992), and (b) the potency of ATP was higher, not lower, there than on the oocyte (Table 1). On the oocyte surface, the bulk concentration of the nucleotides was maintained by superfusion, changing the recording cham- ber volume every 6 s. A recent study (Ziganshin et al., 1995) of the ecto-ATPase activity on Xenopus oocytes has shown that there is little hydrolysis of ATP and 2-MeSATP in the conditions we use, as has also been found for other sources (Welford et al., 1987).

4.3. The high potency of 2-MeSATP

The high absolute value of the physiological potency of 2-MeSATP on this recombinant receptor (ECs0 = 10 nM,

288 J. Simon et al. / European Journal of Pharmacology - Molecular Pharmacology Section 291 (1995) 281 289

n = 6 oocytes) is noteworthy. Such an affinity has not been seen at most peripheral sites of P2v purinoceptors, i.e. on smooth muscle, endothelial and glandular sites (Burnstock, 1993; Dubyak and Fedan, 1991; O'Connor et al., 1991). However it is not unknown, since the same ECs0 of 10 nM has been found (measuring the activation by 2-MeSATP of 1,4,5InsP 3 formation) for a P2¥ purinoceptor on turkey erythrocyte membranes (Boyer et al., 1990). The turkey homologue of the chicken P2w receptor has recently been obtained by Filtz et al. (1994) via polymerase chain reac- tion (using chicken P2vl sequence primers) of turkey brain cDNA, and has only one amino acid difference (threonine- 28 to serine). For 2-MeSATP, those investigators found an ECs0 value of 30.5 _+ 14.8 nM (in an 1,4,5InsP 3 accumula- tion assay in transfected human astrocytoma cells), a value reasonably close to ours in the oocyte. However, for unknown reasons the ratio found for the potencies of 2-MeSATP and ATP, noted above as being high in our oocyte studies at 15, is greater still (37) for the turkey P2¥~ receptor (Filtz et al., 1994).

A higher potency of ATP over ADP, as found in the present study, is also a characteristic of a P2v purinoceptor present on many peripheral tissues (Allen and Burnstock, 1990) and on hepatocytes (Keppens, 1993), but it is not a feature shared by Pzv purinoceptors on glial cells (Pearce et al., 1989; Salter and Hicks, 1994) and endothelial cells (Houston et al., 1987) or those expressed in oocytes in- jected with guinea pig brain mRNA (Foumier et al., 1990). Mixed P2 receptor types undoubtedly occur in many of these situations, which can complicate their analysis. The dose-response relationship for ADP at the recombinant receptor expressed in the oocytes requires discussion, since the data were fitted best by a biphasic curve (Fig. 3). The calculated affinity for ADP from the first phase of the oocyte dose-response curve, where it paralleled (but was displaced to the right of) the curve for ATP, was in good agreement with the K i value of ADP measured on COS-7 cells expressing this P2v~ purinoceptor. The cause of the shallow second phase of the ADP dose-response curve is uncertain, but we can suggest as a possibility that it derives from the presence of an undetected blocking contaminant in the ADP stock solution showing much lower affinity. Filtz et al. (1994) have also found a much lower functional affinity of ADP for the native P2vl receptor on turkey erythrocytes (ECs0:8 /zM) than for the expressed recom- binant turkey P2w receptor (534 nM); those two values approximately correspond to the two phases for ADP in Fig. 3.

4.4. Transductional pathway

ATP acting through the P2Y1 receptor was a potent stimulator of the phospholipase C pathway (Fig. 4A). This agonist effect is shared by dATPaS, used in the radioli- gand binding experiments, as well as ATPaS (which is more active) and ATPyS (Fig. 4B). Receptors which ex-

press strongly in the Xenopus oocyte (as here) are in most cases those which couple to 1,4,5InsP 3 formation. Like- wise, the homologous turkey receptor expressed in astrocy- toma cells (Filtz et al., 1994) stimulates 1,4,5InsP 3 forma- tion. Therefore, the coupling seen here in COS-7 cells was not due to a restricted set of G-proteins in one cell line but suggests that (as is common with P2Y receptors) it is the native transduction pathway of this P2¥1 receptor.

In conclusion, this recombinant brain receptor is a specific P2v purinoceptor subtype, which we have charac- terised in two expression systems. We have used the above-described pharmacological criteria to designate this receptor type as Pzvl. This proposal is in keeping with suggestions made by Abbracchio and Burnstock (1994) to classify all of the metabotropic P2 purinoceptors recog- nised functionally, in a series (so far) of P2vi . . . P z Y 7 . The transiently expressed Pv~ purinoceptor is seen to be cou- pled to the 1,4,5InsP 3 pathway of phospholipase C activa- tion, presumably via either G ~ o r Gaq , which are both known to be expressed in the COS-7 cells used (Wu et al., 1992). The abundant presence of this receptor subtype revealed in the brain, where it shows a discrete expression pattern (Webb et al., 1994), focuses attention on a new role for ATP in slow neurotransmission systems of the brain employing this purinoceptor subtype.

Acknowledgements

J.S. and T.E.W. contributed equally to this work. Elec- trophysiological experiments were carried out by B.F.K. who was assisted, in part, by Dr. Shuyan Wong (on leave from Beijing University, China). T.E.W. is supported by Fisons Pharmaceuticals R&D. B.F.K. is supported by the Wellcome Trust. We thank Dr. Paul Left (Fisons Pharma- ceuticals R&D) for his support of this work.

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